An antigen is a substance causing a specific response in which resistance to a particular foreign substance develops after initial exposure to it. Antigens may be proteins, polysaccharides, nucleic acids, lipids, synthetic polymers and microorganisms, just to name a few. Antigens are characterized by a certain minimum size. Antigens may be as small as 1,000 daltons or as large as millions of daltons.
Upon exposure to an antigen, specific responses develop in a human or animal, one of which is the synthesis and releasing of antibodies from the lymphocytes and plasma cells. Antibodies are specific protein reagents that combine with antigens.
There are five classes of human antibodies (immunoglobulins), namely, IgA, IgD, IgE, IgG and IgM.
An antibody can bind to only a relatively small exposed portion of the surface of the antigen. This small reactive portion is said to be the antigenic determinant (epitope). An antibody is thus constructed to fit a particular exposed arrangement of chemical groups in the antigen, and it will not fit other regions of the antigen's surface. The binding site of an antibody will often "recognize" determinants of closely related structures with at least a partial fit into the site, but in general the tightest combination of antibody and antigen is achieved only when the fit is very good and complete. Accordingly, any synthetic reagent for the detection of antigens or antibodies must have a similar configuration to the corresponding natural antigenic determinant so as to be recognized by and to bind with the antibodies.
The upper limit on the size of antigenic determinants as estimated from studies on linear polypeptides, is about 30.times.17.times.6.5 Angstroms, or a volume of about 3,000 cubic Angstroms. The size of the antigenic sites on proteins is generally of the same magnitude, but these sites may also involve amino acids which are not sequentially arranged, but which are in close proximity due to the folding of the polypeptide chain.
An antigen such as a monomeric protein may contain more than a single antigenic determinant. Because of its limited size, the surface (volume) of the antigenic determinant represents only a minor fragment of the surface of the whole protein.
The amino acid residues corresponding to antigenic determinants contributing the highest proportion of the binding energy between an antigen and an antibody are referred to as the immunodominant group. In viral capsids and complex proteins (polymers), identical or non-identical monomeric units interact in a way that involves part of their total surface. Any antigenic determinants occurring in this part of the monomers (viral structural components) are hidden and become available only upon disaggregation of the polymers. Such antigenic determinants are referred to as cryptotopes.
Antigenic determinants which are available at the surface of both of the polymers and the monomers are referred to as metatopes. The proper assembly of identical or non-identical monomers into polymers may result in the appearance of different antigenic determinants. These antigenic determinants may be comprised of amino acid residues from different monomers, or they may arise from conformational changes in the monomers such as, for example, allosteric transitions. Such antigenic determinants are referred to as neotopes.
The immunoinactivation of virus infectivity, for example, results from the combination of antibodies with surface antigen(s) on the virus particle. Consequently, metatopes and/or neotopes play the essential role in eliciting anti-viral antibodies in immunized humans or animals. If neotopes were absolutely essential, or if they played an immunodominant role, antibodies formed as a result of the immunization with monomers either would completely fail to neutralize the virus or would, perhaps, have low avidity (as measured by neutralization of virus infectivity). Considering the above, it is noted that neotopes may arise not only from the complete assembly of monomers (viral structural components) into a final polymer (viral capsid or envelope), but also from a limited association of a few identical or nonidentical monomers.
Hydrophilic (polar) amino acid side chains usually occur more frequently on the surface of proteins exposed to the surrounding medium, than in a position where they are shielded from the medium. Therefore, such amino acid residues are likely to represent antibody binding sites.
It has been suggested that in the evolution of viruses the most type-specific antigens became located on the outermost parts of the viral structure. Following this concept, neotopes and metatopes should have developed into highly type-specific antigenic determinants, while cryptotopes should be common for a certain group of viruses. In other words, neotopes and metatopes should be part of a variable amino sequence, and cryptotopes, part of a relatively invariable amino sequence within the evolving amino sequences of the constituent polypeptide chains of viral proteins.
The availability of amino acid sequence data (most easily deduced from the determination of the nucleotide sequence of the gene coding for the particular protein antigen) for protein antigens of pathogenic organisms has lead to attempts to identify the amino acid sequence corresponding to the immunodominant determinant, synthesize the corresponding sequence and utilize it as a synthetic vaccine, R. Arnon, "Chemically Defined Antiviral Vaccines", Ann. Rev. Microbiol., 34, 593-618, 1980.
Amino acid sequences can be identified on the basis of the following: (1) by immunological studies on fragments of protein antigens obtained by cleavage with proteolytic enzymes or chemical reagents; (2) by synthesis of a series of consecutive overlapping peptides that represent the entire primary structure of the protein, A. L. Kazim and M. Z. Atassi, "A Novel and Comprehensive Synthetic Approach for the Elucidation of Protein Antigenic Structures", Biochem. J., 191, 261-264, 1980; R. A. Lerner, "Chemically Synthesized Peptides Predicted From the Nucleotide Sequence of the Hepatitis B Virus Genome Elicit Antibodies Reactive With the Native Envelope Protein of Dane Particles", Proc. Natl. Acad. Sci. USA, 78, 3403-3407, 1981; (3) by alterations of antigenicity caused by chemical reagents specifically reacting with distinct amino acid residues, A. R. Neurath and N. Strick, "Localization of a Hepatitis B Surface Antigen Determinant Deduced from Results of Chemical Modifications", J. Virol. Methods, 3, 115-125, 1981; or (4) by determining the region of the highest hydrophilicity within the amino sequence of a protein, T. P. Hopp and K. R, Woods, "Prediction of Protein Antigenic Determinants From Amino Acid Sequences", Proc. Natl. Acad. Sci., 78, 3824-3828, 1981.
Immunodominant antigenic determinants may not necessarily be located on a contiguous amino acid sequence representing the primary structure of a protein, but may be composed of residues brought into proximity by the proper folding of the polypeptide chain. Such folding may be stabilized by disulfide bonds between cysteine residues. In such instances, the antigenic determinants may be mimicked by synthetic peptides composed of amino acid residues not directly linked in the sequence of the protein, C. L. Lee and M. Z. Atassi, "Delineation of the Third Antigenic Site of Lysozyme by Application of a Novel `Surface-Simulation` Synthetic Approach Directly Linking the Conformationally Adjacent Residues Forming the Site", Biochem. J., 159, 89-93, 1976.
Most of the recent studies on synthetic peptides carrying antigenic determinants of viruses or pathogenic microorganisms do not address the issue of whether or not synthetic peptides can protect immunized humans or animals against infection. These studies show, however, that synthetic peptides can either induce antibodies reacting with the "natural" antigen, or conversely that the synthetic peptides can bind at least a portion of antibodies against the "natural" antigen. These studies are not sufficient to show that such synthetic peptides, when used for vaccination, would indeed render humans or animals resistant to infectious agents carrying epitopes (antigen binding sites having a few amino acid residues) homologous with the particular synthetic peptides. In fact, recent reports indicate that antibodies against viral subunits may fail to neutralize the infectivity of the virus, M. M. Hardy and D. M. Moore, "Neutralization of Foot-and Mouth Disease Virus. I. Sensitization of the 140 S Virion by Antibody Also Reactive With the 12 S Protein Subunit", J. Gen. Virol., 55, 415-427, 1981; or that monoclonal antibodies against some viral epitopes may inhibit virus neutralization, R. J. Massey and G. Schochetman, "Viral Epitopes and Monoclonal Antibodies: Isolation of Blocking Antibodies That Inhibit Virus Neutralization", Science, 213, 447-450, 1981.
On the other hand, laboratory animals were successfully immunized against diphtheria toxin using a synthetic oligopeptide in F. Audibert et al., "Active Antitoxic Immunization by a Diphtheria Toxin Synthetic Oligopeptide", Nature, 289, 593-594, 1981, and a synthetic polypeptide elicited antibodies which promoted phagocytosis and the killing of a pathogenic bacterium in E. H. Beachey et al., "Type-Specific Protective Immunity Evoked By Synthetic Peptide of Streptococcus Pyogenes M Protein", Nature, 292, 457-459, 1981.
A synthetic antigen was utilized for the determination of carcinoembryonic antigen levels in sera of cancer patients, R. Arnon et al., "Viroimmunoassay Utilizing a Synthetic Peptide: A Test Equivalent To The Carcinoembryonic Antigen Radioimmunoassay", Isr. J. Med. Sci., 13, 1022-1027, 1977.
A synthetic vaccine comprising a synthetic peptide on a carrier wherein the peptide has a sequence of amino acids corresponding to the sequence of amino acids in a protein, antigen or allergen is described in copending application Ser. No. 223,558, filed Jan. 9, 1981, assigned to the same assignee as the present invention.
Monoclonal antibodies prepared against the natural antigen are described by G. Galfre and C. Milstein, Methods in Enzymology, Vol 73, "Immunochemical Techniques, Part B, Preparation of Monoclonal Antibodies: Strategies and Procedures", 1-45, Academic Press, New York, 1981.
Immunoassays utilizing radiolabeled, enzyme labeled, fluorescent or chemiluminescent substances are described in Methods in Enzymology, Volumes 70, 73 and 74, "Immunochemical Techniques, Parts A, B, C", Academic Press, New York, 1980-1982; U.S. Pat. No. 4,297,273 to Buckler et al.; A. R. Neurath and N. Strick, "Enzyme-Linked Fluorescence Immunoassays Using .beta.-Galactosidase and Antibodies Covalently Bound to Polystyrene Plates", J. Virol. Methods, 1981, 3, 155-165.
The above indicates the need for diagnostic methods relating to the interaction of antibodies (elicited by synthetic peptides) with the corresponding antigens. Such methods would avoid the initial utilization of clinical trials with synthetic vaccines which would be very costly and may potentially involve health risks. The present invention not only satisfies the above described need, but also provides for the utilization of synthetic peptides as diagnostic tools in general for detection of antibodies and antigens.